Very large scale immobilized polymer synthesis using mechanically directed flow paths

ABSTRACT

A method and device for forming large arrays of polymers on a substrate (401). According to a preferred aspect of the invention, the substrate is contacted by a channel block (407) having channels (409) therein. Selected reagents are flowed through the channels, the substrate is rotated by a rotating stage (403), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

This is a Continuation of application Ser. No. 08/246,590, filed May 20,1994, now abandoned, which is a Division of application Ser. No.07/796,243, filed Nov. 22, 1991, U.S. Pat. No. 5,384,261, issued Jan.24, 1995 the entire contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the field of polymer synthesis. Morespecifically, in one embodiment the invention provides an improvedmethod and system for synthesizing arrays of diverse polymer sequences.According to a specific aspect of the invention, a method ofsynthesizing diverse polymer sequences such as peptides oroligonucleotides is provided. The diverse polymer sequences may be used,for example, in screening studies for determination of binding affinity.

Methods of synthesizing desired polymer sequences such as peptidesequences are well known to those of skill in the art. For example, theso-called "Merrifield" solid-phase peptide synthesis has been in commonuse for several years and is described in Merrifield, J. Am. Chem Soc.(1963) 85:2149-2154, incorporated herein by reference for all purposes.Solid-phase peptide synthesis techniques have been extended to providefor the synthesis of several peptide sequences on, for example, a numberof "pins" as described in, for example, Geysen et al., J. Immun. Meth.(1987) 102:259-274, also incorporated herein by reference for allpurposes. Methods of synthesizing oligonucleotides are found in, forexample, Oligonucleotide Synthesis: A Practical Approach, Gait, ed., IRLPress, Oxford (1984), incorporated herein by reference in its entiretyfor all purposes.

Such methods and devices have continued to be limited in the number ofsequences which can be synthesized in a reasonable amount of time. Forexample, Geysen et al. report in the above journal that it has takenapproximately 3 years to synthesize 200,000 peptide sequences. Suchmethods have continued to produce fewer peptide sequences for study thanare often desired.

Accordingly, improved methods of forming large arrays of peptides,oligonucleotides, and other polymer sequences in a short period of timehave been devised.

Of particular note, Pirrung et al., PCT Application No. WO 90/15070 andU.S. application Ser. No. 07/624,120 abandoned in favor of U.S. Ser. No.08/390,027 filed Feb. 16, 1995, both incorporated herein by reference,disclose methods of forming vast arrays of peptides and other polymersequences using, for example, light-directed synthesis techniques. Seealso, Fodor et al., Science (1991) 251:767-777, also incorporated hereinby reference for all purposes.

These techniques have met with substantial success. However, in somecases it is desirable to have alternate/additional methods of formingpolymer sequences which would not utilize, for example, light as anactivator, or which would not utilize light exclusively.

SUMMARY OF THE INVENTION

Methods and devices for synthesizing arrays of diverse polymer sequencessuch as diverse peptides and oligonucleotides are provided by virtue ofthe present invention. According to a preferred embodiment of theinvention, a series of channels or grooves are formed on or adjacent asubstrate. Reagents are selectively flowed through or placed in thechannels or grooves, forming polymers having different monomer sequencesat selected locations on the substrate.

According to the first specific aspect of the invention, a block havinga series of grooves on a surface thereof is utilized. The block isplaced in contact with a derivatized glass or other substrate. In afirst step, a pipettor or other delivery system is used to flow selectedreagents to one or more of a series of apertures connected to thegrooves, or place reagents in the grooves directly, filling the groovesand "striping" the substrate with a first reagent, coupling a firstmonomer thereto. The grooves may in some embodiments thereafter beprovided with additional reagents, providing coupling of additionalmonomers to the first monomer. The block is then translated or rotated,again placed on the substrate, and the process is repeated with a secondreagent, coupling a second monomer to different regions of thesubstrate. The process is repeated until a diverse set of polymers ofdesired sequence and length is formed on the substrate. By virtue of theprocess, a number of polymers having diverse monomer sequences such aspeptides or oligonucleotides are formed on the substrate at knownlocations.

According to the second aspect of the invention, a series ofmicrochannels or microgrooves are formed on a substrate, along with anappropriate array of microvalves. The channels and valves are used toflow selected reagents over a derivatized surface. The microvalves areused to determine which of the channels are opened for any particularcoupling step.

Accordingly, one embodiment of the invention provides a method offorming diverse polymer sequences on a single substrate, the substratecomprising a surface with a plurality of selected regions. The methodincludes the steps of forming a plurality of channels adjacent thesurface, the channels at least partially having a wall thereof definedby a portion of the selected regions; and placing selected reagents inthe channels to synthesize polymer sequences at the portion of theselected regions, the portion of the selected regions comprisingpolymers with a sequence of monomers different from polymers in at leastone other of the selected regions.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a generalized diagram illustrating the invention;

FIG. 2 is a flow chart illustrating the treatment steps performed insynthesizing an array of various polymers;

FIG. 3 is a mapping of a resulting array of polymers;

FIG. 4a is a top view and FIG. 4b is a cross-sectional view of a firstembodiment of a device used to synthesize arrays of polymer sequences;

FIGS. 5a and 5b illustrate alternative arrangements of the grooves in achannel block;

FIGS. 6a and 6b illustrate a microvalve device;

FIGS. 7a and 7b illustrate an alternative embodiment of the invention;

FIG. 8 is a mapping of expected fluorescent intensities with a substrateselectively exposed to fluorescent dye;

FIG. 9 is a mapping of actual fluorescent intensities; and

FIG. 10 is a mapping of intensity versus location with a slide havingYGGFL, GPGFL, pGGFL, and pPGFL synthesized thereon.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Contents

I. Glossary

II. General

III. Details of a First Embodiment

IV. Details of a Second Embodiment

V. Alternative Embodiments

VI. Examples

A. Leak Testing

B. Formation of YGGFL

VII. Conclusion

I. Glossary

The following terms are intended to have the following general meaningsas they are used herein:

1. Ligand: A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, cofactors, drugs (e.g., opiates, etc.) lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies.

2. Monomer: A member of the set of small molecules which are or can bejoined together to form a polymer. The set of monomers includes but isnot restricted to, for example, the set of common L-amino acids, the setof D-amino acids, the set of synthetic and/or natural amino acids, theset of nucleotides and the set of pentoses and hexoses.

The particular ordering of monomers within a polymer is referred toherein as the "sequence" of the polymer. As used herein, monomers refersto any member of a basis set for synthesis of a polymer. For example,dimers of the 20 naturally occurring L-amino acids form a basis set of400 monomers for synthesis of polypeptides. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.Furthermore, each of the sets may include protected members which aremodified after synthesis. The invention is described herein primarilywith regard to the preparation of molecules containing sequences ofmonomers such as amino acids, but could readily be applied in thepreparation of other polymers. Such polymers include, for example, bothlinear and cyclic polymers of nucleic acids, polysaccharides,phospholipids, and peptides having either α-, β-, or ω-amino acids,heteropolymers in which a known drug is covalently bound to any of theabove, polynucleotides, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides, 4polysiloxanes, polyimides, polyacetates, or other polymers which will beapparent upon review of this disclosure. Such polymers are "diverse"when polymers having different monomer sequences are formed at differentpredefined regions of a substrate. Methods of cyclization and polymerreversal of polymers are disclosed in copending application Ser. No.07/796,727, now U.S. Pat. No. 5,242,974, filed on the same date as thepresent application, entitled "POLYMER REVERSAL ON SOLID SURFACES,"incorporated herein by reference for all purposes.

3. Peptide: A polymer in which the monomers are alpha amino acids andwhich are joined together through amide bonds, alternatively referred toas a polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are often two or more amino acid monomerslong, and often more than 20 amino acid monomers long. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

4. Receptor: A molecule that has an affinity for a given ligand.Receptors may be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A"Ligand Receptor Pair" is formed when two macromolecules have combinedthrough molecular recognition to form a complex.

Specific examples of receptors which can be investigated by thisinvention include but are not restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in developing a new class ofantibiotics of particular value would be antibiotics againstopportunistic fungi, protozoa, and those bacteria resistant to theantibiotics in current use.

b) Enzymes: For instance, the binding site of enzymes such as theenzymes responsible for cleaving neurotransmitters; determination ofligands which bind to certain receptors to modulate the action of theenzymes which cleave the different neurotransmitters is useful in thedevelopment of drugs which can be used in the treatment of disorders ofneurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for autoimmunediseases (e.g., by blocking the binding of the "self" antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant. Catalytic polypeptides and others are described in, forexample, PCT Publication No. WO 90/05746, WO 90/05749, and WO 90/05785,which are incorporated herein by reference for all purposes.

f) Hormone receptors: For instance, encompasses the receptors forinsulin and growth hormone. Determination of the ligands which bind withhigh affinity to a receptor is useful in the development of, forexample, an oral replacement of the daily injections which diabeticsmust take to relieve the symptoms of diabetes, and in the other case, areplacement for the scarce human growth hormone which can only beobtained from cadavers or by recombinant DNA technology. Other examplesare the vasoconstrictive hormone receptors; determination of thoseligands which bind to a receptor may lead to the development of drugs tocontrol blood pressure.

g) opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

5. Substrate: A material having a rigid or semi-rigid surface. In manyembodiments, at least one surface of the substrate will be substantiallyflat, although in some embodiments it may be desirable to physicallyseparate synthesis regions for different polymers with, for example,wells, raised regions, etched trenches, or the like. According to otherembodiments, small beads may be provided on the surface which may bereleased upon completion of the synthesis.

6. Channel Block: A material having a plurality of grooves or recessedregions on a surface thereof.

The grooves or recessed regions may take on a variety of geometricconfigurations, including but not limited to stripes, circles,serpentine paths, or the like.

7. Protective Group: A material which is bound to a monomer unit andwhich may be selectively removed therefrom to expose an active site suchas, in the specific example of an amino acid, an amine group.

8. Predefined Region: A predefined region is a localized area on asubstrate which is, was, or is intended to be used for formation of aselected polymer and is otherwise referred to herein in the alternativeas a "selected" region or simply a "region." The predefined region mayhave any convenient shape, e.g., circular, rectangular, elliptical,wedge-shaped, etc. In some embodiments, a predefined region and,therefore, the area upon which each distinct polymer sequence issynthesized is smaller than about 1 cm², more preferably less than 1mm², still more preferably less than 0.5 mm², and in some embodimentsabout 0.125 to 0.5 mm². In most preferred embodiments the regions havean area less than about 10,000 μm² or, more preferably, less than 100μm². Within these regions, the polymer synthesized therein is preferablysynthesized in a substantially pure form.

9. Substantially Pure: A polymer is considered to be "substantiallypure" within a predefined region of a substrate when it exhibitscharacteristics that distinguish it from other predefined regions.Typically, purity will be measured in terms of biological activity orfunction as a result of uniform sequence. Such characteristics willtypically be measured by way of binding with a selected ligand orreceptor. Preferably the region is sufficiently pure such that thepredominant species in the predefined region is the desired sequence.According to preferred aspects of the invention, the polymer is 5% pure,more preferably more than 10% pure, preferably more than 20% pure, morepreferably more than 80% pure, more preferably more than 90% pure, morepreferably more than 95% pure, where purity for this purpose refers tothe ratio of the number of ligand molecules formed in a predefinedregion having a desired sequence to the total number of molecules formedin the predefined region.

II. General

The present invention provides for the synthesis of arrays of largenumbers of different polymer sequences. According to a preferredembodiment of the invention, the invention provides for the synthesis ofan array of different peptides in selected regions of a substrate. Suchsubstrates having the diverse sequences formed thereon may be used in,for example, screening studies to evaluate their interaction withreceptors such as antibodies. For example, in preferred embodiments theinvention provides for screening of peptides to determine which if anyof a diverse set of peptides has strong binding affinity with a receptorand, in most preferred embodiments to determine the relative bindingaffinity of various peptides with a receptor of interest such as anantibody.

Such diverse polymer sequences are preferably synthesized on a singlesubstrate. By synthesizing the diverse polymer sequences on a singlesubstrate, processing of the sequences to evaluate theircharacteristics, such as relative binding affinity, is more easilyconducted. By way of example, when a variety of peptide sequences are tobe evaluated to determine their relative binding affinity to a receptor,the entire substrate and, therefore, all or a group of the polymersequences may be exposed to an appropriately labelled receptor andevaluated simultaneously.

The diverse polymer sequences are synthesized at selected regions of asubstrate by forming flow channels on a surface of the substrate throughwhich appropriate reagents flow or in which appropriate reagents areplaced. For example, assume a monomer "A" is to be bound to thesubstrate in a first group of selected regions. If necessary, all orpart of the surface of the substrate in all or a part of the selectedregions is activated for binding by, for example, flowing appropriatereagents through all or part of the channels, or by washing the entiresubstrate with appropriate reagents. After placement of a channel blockon the surface of the substrate, a reagent having the monomer A flowsthrough or is placed in all or a part of the channel(s). The channelsprovide fluid contact to the first selected regions, thereby binding themonomer A on the substrate directly or indirectly (via a linker) in thefirst selected regions.

Thereafter, a monomer B is coupled to second selected regions, some ofwhich may be included among the first selected regions. The secondselected regions will be in fluid contact with a second flow channel(s)through translation, rotation, or replacement of the channel block onthe surface of the substrate; through opening or closing a selectedvalve; or through deposition of a layer of photoresist. If necessary, astep is performed for activating at least the second regions.Thereafter, the monomer B is flowed through or placed in the second flowchannel(s), binding monomer B at the second selected locations. In thisparticular example, the resulting sequences bound to the substrate atthis stage of processing will be, for example, A, B, and AB. The processis repeated to form a vast array of sequences of desired length at knownlocations on the substrate.

Various embodiments of the invention will provide for other methods offorming channels or otherwise protecting a portion of the surface of thesubstrate. For example, according to some embodiments a protectivecoating such as a hydrophilic coating is utilized over portions of thesubstrate to be protected, sometimes in combination with the use ofvarious wetting materials and the like in other regions.

FIG. 1 illustrates an example of the invention. In this particularexample, monomers and dimers of the monomer group A, B, C, and D are tobe bound at selected regions of the substrate. The substrate may bebiological, nonbiological, organic, inorganic, or a combination of anyof these, existing as particles, strands, precipitates, gels, sheets,tubing, spheres, containers, capillaries, pads, slices, films, plates,slides, etc. The substrate may have any convenient shape, such as adisc, square, sphere, circle, etc. The substrate is preferably flat butmay take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whichthe synthesis takes place.

The substrate and its surface form a support on which to carry out thereactions described herein. These monomers are bound using first flowchannel paths x₁, x₂, X₃, and x₄ which are formed or placed on oradjacent the substrate in a first orientation, and second flow channelpaths y₁, y₂, y₃, and y₄ which are formed or placed on or adjacent thesubstrate in a second orientation. The second flow channel pathsintersect at least a part of the first flow channel paths. The flowchannels are formed according to techniques which are described ingreater detail elsewhere herein.

Initially the substrate is subjected to one or more preliminarytreatments such as, for example, cleaning and the optional placement of"linker" molecules on the surface thereof. The substrate may also beprovided with various active groups, common monomer sequences which willform a part of the polymers, or the like.

Thereafter, in a first coupling step, one or more of the flow channelsare provided with the first monomer A, which binds through covalentbonds or otherwise to the substrate (directly or indirectly) where theflow channel contacts the substrate. In the particular example shown inFIG. 1, the flow channels x₁ and x₂ are utilized, binding the monomer Ato the substrate along the entire length of the substrate adjacent tothe x₁ and x₂ channels. It will be understood that each coupling stepmay in some embodiments be composed of a variety of substeps. Forexample, each coupling step may include one or more substeps forwashing, chemical activation, or the like.

Thereafter or concurrently therewith, as shown in FIG. 2, a secondmonomer B is provided to selected flow channels and the monomer B bindsto the substrate where the second flow channels provide contacttherewith. In the particular example shown in FIG. 2, monomer B is boundalong channels x₃ and x₄. When the monomers A and B flow through theirrespective flow channels simultaneously, only a single process step isrequired to perform two coupling steps simultaneously. As used herein, a"process step" refers to the injection of one or more channels with oneor more reagents. A "coupling step" refers to the addition of a monomerin a polymer.

Processing thereafter continues in a similar manner with monomers C andD in the manner shown in the flow diagram of FIG. 2, with monomer Cbeing bound in the flow channels y₁ and y₂, and D being bound in theflow channels y₃ and y₄. Preferably, monomers C and D are directedthrough the flow channels y₁ to y₄ simultaneously whereby two couplingsteps are performed with a single process step. Light regions in FIG. 1indicate the intersections of the resulting flow paths.

FIG. 3 illustrates the mapping of sequences formed using the aboveillustrated steps. As shown therein, the sequences A, B, C, D, AD, BD,AC, and BC have been formed using only two process steps. Accordingly,it is seen that the process provides for the synthesis of vast arrays ofpolymer sequences using only a relatively few process steps. By way offurther example, it is necessary to use only two process steps to formall of the 4² =16 dimers of a four-monomer basis set. By way of furtherexample, to form all 4⁸ octomers of a four-monomer basis set, it isnecessary to provide only 256 flow channels oriented in the "x"direction, and 256 flow channels oriented in the "y" direction, with atotal of eight coupling steps. Accordingly, it is seen that the presentinvention provides a highly efficient method of performing synthesis ofdiverse polymers.

III. Details of a First Embodiment

FIGS. 4a and 4b illustrate details of a first embodiment of a deviceused for performing the synthesis steps described above. In particular,FIG. 4a illustrates the device in top view, while FIG. 4b illustratesthe device in cross-sectional side view. In the particular embodimentshown in FIG. 4, the device is used to synthesize polymer sequences onsubstrate 401. substrate 401 is coupled to a rotating stage 403 andremovably held by clamp 405 to channel block 407. Channel block 407 hasetched therein a plurality of channels 409 in the form of stripestherein. Each channel is provided with a flow inlet 411 and an outlet413. A vacuum source 415 is applied to one or more of the outlets 413,while a pipettor 417 is slidably mounted on arm 419 to deliver selectedreagents from reservoir(s) 421 to a selected one of the flow inlets 411.

It will be recognized that in some embodiments the channel block willnot be utilized. Instead, in some embodiments, small "strips" of reagentare applied to the substrate by, for example, striping the substrate orchannels therein with a pipettor. According to other embodiments thechannels will be formed by depositing an electron or photoresist such asthose used extensively in the semiconductor industry. Such materialsinclude polymethyl methacrylate (PMMA) and its derivatives, and electronbeam resists such as poly(olefin sulfones) and the like (more fullydescribed in Ghandi, "VLSI Fabrication Principles," Wiley (1983) Chapter10, incorporated herein by reference in its entirety for all purposes).According to these embodiments, a resist is deposited, selectivelyexposed, and etched, leaving a portion of the substrate exposed forcoupling. These steps of depositing resist, selectively removing resistand monomer coupling are repeated to form polymers of desired sequenceat desired locations.

In preferred embodiments, the substrate is conventional glass, pyrex,quartz, any one of a variety of polymeric materials, or the like. Ofcourse, the substrate may be made from any one of a variety of materialssuch as silicon, polystyrene, polycarbonate, or the like. In preferredembodiments the channel block is made of polychlorotrifluorethylene,such as material known under the trade name Kel-F™ 80(chlorotrifluoroethylenevinylidene fluoride) made by 3M, although a widevariety of materials such as silicon, polystyrene, polycarbonate, glass,elastomers such as Kalrez made by DuPont, various ceramics, stainlesssteel, or the like may be utilized.

The channels in the channel block are preferably made by machining,compression molding, injection molding, lithography, laser cutting, orthe like depending upon the material of interest. In preferredembodiments the raised portions of the channels in the channel block aretreated by lapping with lapping film (0.3 μm grit). Such smooth surfacesprovide good seals to the substrate without the use of a sealant and,therefore, without the possibility of leaving sealant material on thesubstrate when rotating the channel block. Preferably, all operationsare conducted at substantially ambient temperatures and pressures.

In operation, the surface of the substrate is appropriately treated bycleaning with, for example, organic solvents, methylene chloride,dimethylformamide (DMF), ethyl alcohol, or the like. Optionally, thesubstrate may be provided with appropriate linker molecules on thesurface thereof. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing from 2-10 monomers ormore, diamines, diacids, amino acids, or combinations thereof.Thereafter, the surface is provided with protected surface active groupssuch as t-butyloxycarbonyl (TBOC) or fluoroenylmethyloxycarbonyl (FMOC)protected amino acids. Such techniques are well known to those of skillin the art.

Thereafter, the channel block and the substrate are brought into contactforming fluid-tight channels bounded by the grooves in the channel blockand the substrate. When the channel block and the substrate are incontact, a protective group removal agent is, thereafter, directedthrough a first selected channel or group of channels by placing thepipettor on the flow inlet of the selected channel and, optionally, thevacuum source on the outlet of the channel. In the case of, for example,TBOC protected amino acids, this protective group removal agent may be,for example, trifluoroacetic acid (TFA). This step is optionallyfollowed by steps of washing to remove excess TFA with, for example,dichloromethane (DCM).

Thereafter, a first amino acid or other monomer A is directed throughthe first selected flow channel. Preferably this first amino acid isalso provided with an appropriate protective group such as TBOC, FMOC,6-nitroveratryloxycarbonyl (NVOC), or the like. This step is alsofollowed by appropriate washing steps. These steps ofdeprotection/coupling are concurrently with or thereafter repeated foradditional channels parallel to the first channel(s) which are to beprovided with the same or different monomers.

Thereafter, the substrate and the channel block are separated and,optionally, the entire substrate is washed with an appropriate materialto remove any unwanted materials from the points where the channelscontact the substrate.

The substrate and/or block is then, optionally, washed and translatedand/or rotated with the stage. In preferred embodiments, the substrateis rotated 90 degrees from its original position, although someembodiments may provide for greater or less rotation, such as from 0 to180 degrees. When the channel block is rotated, it may simultaneously betranslated with respect to the substrate. By "translated" it is intendedto mean any relative motion of the substrate and/or channel block, while"rotation" is intended to refer to rotation of the substrate and/orchannel block about an axis perpendicular to the substrate and/orchannel block. According to some embodiments the relative rotation is atdifferent angles for different stages of the synthesis.

The steps of deprotection, and coupling of amino acids or other monomersis then repeated, resulting in the formation of an array of polymers onthe surface of the substrate. For example, a monomer B may be directedthrough selected flow channels, providing the polymer AB atintersections of the channels formed by the channel block in the firstposition with the channels formed by the channel block after 90-degreerotation.

It will be recognized that while rotation of the channel block isprovided according to preferred embodiments of the invention, suchrotation is not required. For example, by simply flowing differentreagents through the channels, polymers having different monomersequences may be formed. Merely by way of a specific example, a portionof the channels may be filled with monomer "A," and a portion filledwith monomer "B" in a first coupling step. All or a portion of the firstchannels are then filled with a monomer "C," and all or a portion of thesecond channels are filled with a monomer "D," forming the sequences ABand CD. Such steps could be used to form 100 sequences using a basis setof 10 monomers with a 100-groove channel block.

While linear grooves are shown herein in the preferred aspects of theinvention, other embodiments of the invention will provide for circularrings or other shapes such as circular rings with radial grooves runningbetween selected rings. According to some embodiments, channel blockswith different geometric configurations will be used from one step tothe next, such as circular rings in one step and linear stripes in thenext. FIG. 5a illustrates one of the possible arrangements in which thechannels 409 are arranged in a serpentine arrangement in the channelblock 407. Through appropriate translation and/or rotation of thechannel block, polymers of desired monomer sequence are formed at theintersection of the channels during successive polymer additions, suchas at location 501, where the intersection of a previous or subsequentset of channels is shown in dashed lines. FIG. 5b illustrates anotherarrangement in which channels (in this case without flow paths 413) areprovided in a linear arrangement, with groups 503 and 505 located inadjacent regions of the substrate and extending only a portion of thesubstrate length.

In some embodiments of the invention, the various reagents, such asthose containing the various monomers, are not pumped through the flowpath 413.

Instead, the reagent is placed in one of the grooves, such as thegrooves 409 shown in FIG. 5b, filling the groove. The substrate is thenplaced on top of the channel block, and the exposed portions of thesubstrate are permitted to react with the materials in the grooves.

In preferred embodiments, the channels are of the same width as theraised regions between the channels.

According to these embodiments, the substrate may then be movedlaterally by one channel width or an integer multiple of a channelwidth, permitting reaction with and placement of monomers on the regionsbetween the channels in a previous coupling step. Thereafter, thesubstrate or channel block will be rotated for the next series ofcoupling steps.

Through avoidance of the placement of substantial pressure on thesubstrate by the channel block in this manner, damage to theseintervening regions may be avoided. Also, such methods will be resistantto problems created by "dead spots" in the flow channels when reagentsare pumped through the channels. According to these embodiments, thearray of synthesized polymers can easily cover the entire substrate.Such embodiments may simultaneously couple up to, e.g., 20 peptides in asingle step.

In preferred embodiments, the process is repeated to provide more than10 different polymer sequences on the surface of the substrate. In morepreferred embodiments, the process is repeated to provide more than 10²,10³, 10⁴, 10⁵, or more polymer sequences on a single substrate. In someembodiments the process is repeated to provide polymers with as few astwo monomers, although the process may be readily adapted to formpolymers having 3, 4, 5, 6, 10, 15, 20, 30, 40, 50, 75, 100 or moremonomers therein.

According to preferred embodiments, the array of polymer sequences isutilized in one or more of a variety of screening processes, one ofwhich is described in copending application Serial No. 07/796,947 nowU.S. Pat. No. 5,242,974 filed on the same day as the present applicationand incorporated herein by reference for all purposes. For example,according to one embodiment, the substrate is then exposed to a receptorof interest such as an enzyme or antibody. According to preferredembodiments, the receptor is labelled with fluorescein, or otherwiselabelled, so as to provide for easy detection of the location at whichthe receptor binds. According to some embodiments, the channel block isused to direct solutions containing a receptor over a synthesized arrayof polymers. For example, according to some embodiments the channelblock is used to direct receptor solutions having different receptorconcentrations over regions of the substrate.

According to most preferred embodiments, amplification of the signalprovided by way of fluorescein labelling is provided by exposing thesubstrate to the antibody of interest, and then exposing the substrateto a labelled material which is complementary to the antibody ofinterest and which preferably binds at multiple locations of theantibody of interest. For example, in one specific embodiment, if amouse antibody is to be studied, a labelled second antibody may beexposed to the substrate which is, for example, goat antimouse. Suchtechniques are described in copending application Serial No. 08/390,027,previously incorporated herein by reference.

IV. Details of a Second Embodiment

According to some embodiments of the invention, microvalve structuresare used to form channels along selected flow paths on the substrate.According to these embodiments, an array of microvalves are formed andoperated by an overlying or underlying array of electrodes which areused to energize selected valves to open and close such valves.

FIG. 6 illustrates such a structure, FIG. 6a illustrating the system inend view cross-section and FIG. 6b illustrating the system in top view.It will be recognized that the structure shown therein provides for onlytwo synthesis chambers for the purpose of clarity, but in mostembodiments a far greater number of chambers will be provided.Microvalves are discussed in detail in, for example, Zdeblick, U.S. Pat.No. 4,966,646, and Knutti, "Advanced Silicon Microstructures," ASICTConference (1989), both incorporated herein by reference for allpurposes.

As shown therein, a substrate 602 is provided with a plurality ofchannels 604 formed using photolithographic, or other relatedtechniques. The channels lead up to a synthesis chamber 606. At the endof each channel is valve structure 608. As shown in FIG. 6, the channelslead up to the chambers, but may be isolated from the chambers by thevalves. Multiple valves may be provided for each chamber. In theparticular structure shown in FIG. 6, the right valve on the leftchamber and the left valve on the right chamber are open while theremaining valves are closed.

Accordingly, if reagent is delivered to the top of the substrate, itwill flow through the open channel to and through the chamber on theleft, but not the one on the right. Accordingly, coupling steps may beconducted on the chamber with selected reagents directed to selectedchambers, using the techniques discussed above.

According to some embodiments, a valve is supplied on one side of thechamber 606, but the valve on the opposite side is replaced by asemi-permeable membrane. According to these embodiments it becomespossible to flow a selected reagent into the chamber 606 and,thereafter, flow another selected reagent through the flow channeladjacent the semi-permeable membrane. The semi-permeable membrane willpermit a portion of the material on one side or the other to passthrough the membrane. Such embodiments will be useful in, for example,cell studies.

Screening will be performed by, for example, separating or cutting twohalves of the device, enabling screening by, for example, contactingwith a fluorescein labelled antibody, or the like followed byphotodetection.

V. Alternative Embodiments

FIGS. 7a and 7b illustrate one alternative embodiment of the inventionwhich combines the mechanical polymer synthesis techniques disclosedherein with light-directed synthesis techniques. According to theseembodiments, a substrate 401 is irradiated in selected regions, shown asthe non-striped regions in FIG. 7a. The surface of the substrate isprovided with photoremovable groups in accordance with copendingapplication Ser. No. 08/390,027, previously incorporated by referenceon, for example, amine groups in the specific case of peptide synthesis.During this step regions 701, 702, and 703 of the substrate, amongothers, are deprotected, leaving remaining regions of the substrateprotected by photoremovable groups such as NVOC. According to a specificembodiment of the invention the widths of the irradiated regions equalthe widths of the protected regions of the substrate.

Thereafter, as shown in FIG. 7b the substrate is contacted with achannel block 407. In the particular embodiment shown in FIG. 7b, thechannels 704, 705, and 707 are aligned with the regions 701, 702, and703, respectively, on the substrate 401. As will be apparent, specificembodiments of the invention provide for irradiated regions and channelsin the form of stripes, which are aligned during this step. Otherembodiments, however, will provide for other shapes of irradiatedregions and channels, and other relative orientations of the irradiatedregions and channels. The channel block and substrate will be alignedwith, for example, an alignment mark placed on both the substrate andthe channel block. The substrate may be placed on the channel blockwith, for example, a vacuum tip.

Thereafter, a selected reagent is flowed through or placed in thechannels in the channel block for coupling to the regions which havepreviously been exposed to light. As with the first embodiment, thesubstrate may be placed in contact with a prefilled channel block insome embodiments to avoid compression of the channel block to thesubstrate and dead spots during pumping. According to preferred aspectsof the invention, a different reagent flows through each of the channels701, 702, and 703 such as, for example, a reagent containing monomers A,B, and C. The process may then, optionally, involve a second couplingstep in which the substrate is translated by, e.g., one channel width,to provide coupling of a monomer in the regions between the originalchannels.

Thereafter, the process of directed irradiation by light, followed bycoupling with the channel block is repeated at the previously unexposedregions. The process is then preferably repeated again, with the stripesof the mask and the channel block rotated at, for example, 90 degrees.The coupling steps will provide for the formation of polymers havingdiverse monomer sequences at selected regions of the substrate throughappropriate translation of the mask and substrate, and throughappropriate mask selection.

It is seen that through a combination of the light-directed techniquesand the mechanical flow channel techniques disclosed herein, greaterefficiency in forming diverse sequences is achieved since multiplemonomers are coupled in a single irradiation/coupling step.

VI. Examples

A. Leak Testing

An initial experiment was conducted using the device described in thefirst embodiment to assure that materials could be delivered to selectedlocations of a substrate and be prevented from contacting other areas.Additionally, the experiment was used to demonstrate that reagents couldbe delivered in a uniform manner.

Accordingly, a flat piece of conventional glass having dimensions ofabout 42 mm×42 mm was derivatized with aminopropyltriethoxysilane. Theentire slide was deprotected and washed using conventional techniques. Afluorescein marker of fluoresceinisothiocyanate (FITC) was then injectedinto flow channels formed when a block of KelF™ 81 with 10 channels of 1mm depth and 1 mm width were brought into contact with the substrate.The fluorescein marker was in a solution of DMF and flowed through thechannels by injecting the material into the groove with a manual pipet.

Fluorescein dye was similarly injected into every other channel in theblock, the block was rotated, and the process was repeated. The expectedresulting plot of fluorescent intensity versus location is schematicallyillustrated in FIG. 8. Dark regions are shown at the intersections ofthe vertical and horizontal stripes, while lighter grey atnon-intersecting regions of the stripes. The dark grey regions indicateexpected regions of high dye concentration, while the light regionsindicate regions of expected lower dye concentration.

FIG. 9 is a mapping of fluorescence intensity of a portion of an actualslide, with intensity data gathered according to the methods ofcopending application Ser. No. 08/390,027, previously incorporated byreference. The results agree closely with the expected results,exhibiting high fluorescence intensity at the intersection of thechannels (about 50% higher than non-intersecting regions of thestripes), and lower fluorescence intensity at other regions of thechannels. Regions which were not exposed to fluorescence dye show littleactivity, indicating a good signal-to-noise ratio. Intersections havefluorescence intensity about 9× as high as background. Also, regionswithin the channels show low variation in fluorescence intensity,indicating that the regions are being evenly treated within thechannels.

B. Formation of YGGFL

The system was used to synthesize four distinct compounds: YGGFL (SEQ.ID NO:1), YpGFL (SEQ. ID NO:2), pGGFL (SEQ. ID NO:3) and ppGFL. Anentire glass substrate was derivatized with TBOC-protectedaminopropyltriethoxysilane, deprotected with TFA, coated withFMOC-protected caproic acid (a linker), deprotected with piperidine, andcoated with FMOC-protected Glycine-Phenylalanine-Leucine (GFL).

This FMOC-GFL-coated slide was sealed to the channel block, and all 10grooves were deprotected with piperidine in DMF. After washing thegrooves, FMOC Glycine (G) was injected in the odd grooves, and FMOCd-Proline (p) was injected in the even grooves. After a two-hourcoupling time, using standard coupling chemistry, all grooves werewashed with DMF. The grooves were vacuum dried, the block removed androtated 90 degrees. After resealing, all grooves were deprotected withpiperidine in DMF and washed. FMOC Tyrosine (Y) was injected in the oddgrooves, and FMOC p in the even grooves. After coupling the grooves werewashed and vacuum dried. Accordingly, 25 regions of each of thecompounds YGGFL, YpGFL, PGGFL, and ppGFL were synthesized on thesubstrate. The substrate was removed and stained with FITC-labelledantibodies (Herz antibody 3E7).

A section of the resulting slide illustrating fluorescence intensity isshown in FIG. 10. White squares are in locations of YGGFL. The darkestregions are pGGFL and ppGFL. The YGGFL sites were the most intense,followed by the YpGFL sites. The pGGFL and ppGFL intensities were nearbackground levels, consistent with expected results with the Herzantibody.

Quantitative analysis of the results show overall intensity ratios forYGGFL:YpGFL:pGGFL:ppGFL as 1.7:1.5:1.1:1.0. However, since there is alarge standard deviation on the YGGFL and YpGFL, comparing all the siteswith each other may not accurately represent the actual contrasts.Comparing the intensities of sites within the same "stripe" gives largercontrasts, although they remain on the order of 2:1.

VII. Conclusion

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example avariety of substrates, receptors, ligands, and other materials may beused without departing from the scope of the invention. The scope of theinvention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TyrGlyGlyPheLeu    15    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TyrGlyPheLeu    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GlyGlyPheLeu    1    __________________________________________________________________________

What is claimed is:
 1. A method of forming an oligonucleotide arrays forhybridization comprising the steps of:a) contacting a substrate with achannel block in a first orientation, said channel block comprising asubstantially planar member with a plurality of recessed regions andraised walls on a surface thereof, said recessed regions formingchannels, said raised wall effective to produce substantiallyfluid-tight seals between said raised walls and said substrate upon saidcontacting to define thereby a first plurality of flow channels betweensaid channel block and said substrate, said first flow channels dividingsaid substrate surface into at least a first portion and a secondportion; b) flowing at least a first nucleotide through at least one ofsaid first flow channels; and covalently coupling said first nucleotideto said first portion of said substrate surface, said first nucleotidecomprising a reactive group protected by a protecting group, c) flowingat least a second nucleotide through at least one of said firstchannels, and covalently coupling said second nucleotide to said secondportion of said substrate surface, said second nucleotide comprising areactive group protected by a protecting group; d) translating saidchannel block relative to said substrate and contacting said substratewith said channel block in a second orientation to produce substantiallyfluid-tight seals between said raised walls and said substrate upon saidcontacting to define thereby a second plurality of flow channels betweensaid channel block and said substrate, said second flow channelsdividing said substrate surface into at least a third portion and forthportion, said third and fourth portions each having at least oneinteraction with each of said first and second portions of saidsubstrate surface; e) removing said protective group from at least aportion of said first or second nucleotides to form a first set ofdeprotected first or second nucleotides and flowing a third nucleotidethrough at least one of said second flow channels, covalently couplingsaid third nucleotide to at least a portion of said fist set ofdeprotected fist or second nucleotides where said first and secondportions of said substrate surface intersect with said third portion ofsaid substrate surface to form first and second oligonucleotides; and f)removing said protective group from at least a portion of said first andsecond nucleotides to form a second set of deprotected first or secondnucleotides and flowing a fourth nucleotide through at least one of saidsecond flow channels, covalently coupling said fourth nucleotide to atleast a portion of said second set of deprotected first or secondnucleotides where said fourth portion of said substrate surfaceintersects with said first and second portions of said substrate surfaceto form third and fourth oligonucleotides.
 2. The method as recited inclaim 1 wherein at least 10 different oligonucleotides are formed onsaid surface.
 3. The method as recited in claim 1 wherein at least 100different oligonucleotides are formed on said surface.
 4. The method asrecited in claim 1 wherein at least 1,000 different oligonucleotides areformed on said surface.
 5. The method as recited in claim 1 wherein atleast 10,000 different oligonucleotides are formed on said surface. 6.The method as recited in claim 1 wherein at least 100,000 differentoligonucleotides are formed on said surface.
 7. The method as recited inclaim 1 wherein each different oligonucleotide is in a region having anarea of less than about 1 cm².
 8. The method as recited in claim 1wherein each different oligonucleotide is in a region having an area ofless than about 1 mm².
 9. The method as recited in claim 8 wherein eachdifferent oligonucleotide is in a region having an area of less thanabout 10,000 microns².
 10. The method as recited in claim 1 wherein thesurface of said substrate comprises active groups capable of forming acovalent bond with nucleotides, said active groups protected withphotoremovable groups, and wherein the step of forming a first pluralityof flow channels is preceded or followed by a step of irradiatingportions of said substrate with a light source shown through a maskwhereby said photoremovable groups are removed from at least some of theactive groups on said substrate surface in order for at least said firstnucleotide to covalently bind thereto.
 11. The method as recited inclaim 10 wherein said irradiated portions are in the form of stripes,and wherein said step of forming said first plurality of flow channelscomprises forming said first flow channels along a path of said stripes,and wherein different reagents are placed in at least a portion of saidfirst flow channels, said reagents selected from the group consisting ofnucleotides, wash solutions and protective group removal agents.
 12. Themethod as recited in claim 1 wherein each said step of flowing saidfirst, second, third or fourth nucleotide through its respective saidfirst or second flow channels comprises:placing a pipet in fluidcommunication with said first or second flow channel; and injecting saidfirst, second, third or fourth nucleotide from said pipet through itsrespective said first or second flow channel.
 13. The method as recitedin claim 12 wherein said step of placing the pipet in fluidcommunication with said first or second flow channel includes placingsaid pipet in contact with an orifice on a side of said substrate. 14.The method as recited in claim 12 wherein said step of placing the pipetin fluid communication with said first or second flow channel is a stepof placing a plurality of pipettes in fluid communication with aplurality of said first or second flow channels and flowing reagentsselected from the group consisting of said first, second, third andfourth nucleotides, wash solutions and protective group removal agentsthrough at least two of said first or second flow channels.
 15. A methodof forming an array of diverse oligonucleotides at known locations on asingle substrate, the substrate comprising a surface with a plurality ofselected regions, the method comprising:(1) placing a synthesis chamberadjacent the surface enclosing a first selected region of the surface;(2) placing a first selected nucleotide in the synthesis chamber underconditions such that the first nucleotide attaches to the substratewithin the first selected region; (3) translating the synthesis chamberrelative to the substrate such that the synthesis chamber encloses asecond selected region of the surface, part of which is included in thefirst selected region and part of which is not; (4) placing a secondselected nucleotide in the synthesis chamber under conditions such thatthe second selected nucleotide attaches to the substrate or to the firstnucleotide; (5) further translating the synthesis chamber relative tothe substrate such that the synthesis chamber encloses a furtherselected region of the surface, part of which is included in a previousselected region and part of which is not; (6) placing a further selectednucleotide in the synthesis chamber under conditions such that thefurther selected nucleotide attaches to the substrate or to a previousnucleotide attached to the substrate; and (7) repeating steps (5) and(6) until at least 10 diverse oligonucleotides are formed on thesubstrate.
 16. The method of claim 15, wherein steps (5) and (6) arerepeated until at least 100 diverse oligonucleotides are formed on thesubstrate.
 17. The method of claim 16, wherein steps (5) and (6) arerepeated until at least 1000 diverse oligonucleotides are formed on thesubstrate.
 18. The method of claim 15, wherein the diverseoligonucleotides are at least 5 mers.
 19. The method of claim 18,wherein the diverse oligonucleotides are at least 15 mers.
 20. Themethod of claim 15, further comprising the step of screening the diverseoligonucleotides for complementarity to a polynucleotide.